GB2155936A - Cyclosporins - Google Patents

Description

1 r 151

SPECIFICATION

Novel cyclosporins GB 2 155 936 A 1 The present invention relates to novel cyclosporins, processes for their production, their use as pharmaceuticals and pharmaceutical compositions comprising them.

The cyclosporins comprise a class of structurally distinctive, cyclic, poly-N -m ethyl ated undecapeptides commonly possessing pharmacological, in particular immunosuppressive, anti-inflammatory and antiparasitic activity. The first of the cyclosporins to be isolated and the "parent" compound of the class, was the 10 naturally occurring fungal metabolite Cyclosporine, also known as cyclosporin A, of formula A MeBmt-QAbu-Sar-MeLeu-Val-MeLeu-Ala-(D)Aia-MeLeu-MeLeu-MeVaI 1 2 3 4 5 6 7 8 9 10 111 (A) wherein -MeBmt- represents the N-methyi-(4R)-4-but-2E-en-l-yi-4-methyl- (L)threonyI residue of formula B CH3 20 1 1 CH2 1 30 c HO (R) CH (B) \ CH /(R)\" CH13 1 -N-CH-CO- 35 1 (S) CH3 40 in which -x-y- is -CH=CH- (trans).

Since the original discovery of Cyclosporine a wide variety of naturally occurring cyclosporins have been isolated and identified and many further non-natural cyclosporins have been prepared by total- or semi-synthetic means or by the application of modified culture techniques. The class comprised by the cyclosporins is thus now substantial and includes for example the naturally occurring cyclosporins A through Z (c.f. Kobel et al. European Journal of applied Microbiology and Biotechnology 14, 237 240 (1982) and poster presented by Traber et al., 24th. Interscience Conference on Antimicrobial Agents and Chemotherpy, Washington, October 8 - 10,(1984)1; as well as various non- natural or artificial cyclosporins, including dihydro-cyclosporins (in which the group -x-y- of the -MeBmtresidue - see formula B above - is saturated, e.g. as disclosed in US Patents Nos. 4,108,985; 4,210,581 and 4,220,641), cyclosporins in which 50 the -MeBmt- residue is present in isomeric or N-desmethyl form [c.f, European patent no. 0 034 567 and "Cyclosporin A", Proc. internat. Conference on Cyclosporin A, Cambridge (U.K.) September 1981, Ed. D.J.G. White, Elsevier Press (1982) - both describing the total-synthetic method for the production of cyclosporins developed by R. Wenger] and cyclosporins in which incorporation of variant amino acids at specific 55 positions within the peptide sequence is effected (c.f. European Patent no. 0 056 782). Examples of such cyclosporins as disclosed in the above art references include e.g. [Thr]2 -, [Val]'-, [Nva 12- and [Nvaj2[Nvall- cyclosporine (also known as cyclosporins C, D, G and M respectively), [DihydroMeBmtl'-[Va 1]2- Cyclosporine (also known as dihydrocyclosporin D) and [(D)Serl'- and [DihydroMeBmtll-[(D)-Serl'-Cyclosporine. 60 [In accordance with now conventional nomenclature forthe cyclosporins, these are defined throughout the present specification and claims by reference to the structure of Cyclosporine (i.e. cyclosporin A). This is done by first indicating those residues in the molecule which differ from those present in Cyclosporine and then applying the term "Cyclosporine" to characterise the remaining residues which are identical to those present in Cyclosporine. At the same time the term -dihydro-MeBmt- is employed to designate the residue of formula B above in which -x-y- is -CH2-CH2. Thus [Dihydro-MeBmt]'- [Vall'-Cyclosporine is the 65 2 GB 2 155 936 A cyclosporin having the sequence shown in formula A, but in which -MeBmt[formula B, -x-y- = -CH=CH- (trans)] atthe 1-position is replaced by dihydro-MeBmt- [formula B, -x-y- = -CH2_CH2_1 and -aAbu- at the 2position is replaced by -Val. Similarly [(D)Serl'-Cylosporine is the cyclosporin having the sequence shown in formula A, but in which -(D)Alaat the 8-position is replaced by -(D)Ser.

In addition, amino acid residues referred to by abbreviation, e.g. -Ala-, -MeVal- etc... are, in accordance with conventional practice, to be understood as having the (L)-conf ig u ration unless otherwise indicated. Residue abbreviations preceded by "Me", as in the case of -MeLeurepresent N-methylated residues. The individual residues of the cyclosporin molecule are numbered, as in the art, clockwise and starting with the residue -MeBmt- or -dihydro-MeBmt- in position 1. The same numerical sequence is employed 10 throughout the present specification and claims].

In accordance with the present invention it has now been found that novel cyclosporins may be obtained having pharmaceutical utility, in which the residue at the 8-position comprises an acyloxy a-amino acid residue having the (D) -conf igu ration.

Accordingly, in its broadest aspect, the present invention provides: a cyclosporin wherein the amino acid residue at the 8-position is a (D)acyloxy-a-amino acid residue, i.e. the residue of an a-amino acid of the (D)-series wherein the side chain attaching to the a-carbon atom is acyloxy-substituted.

Preferably the amino acid residue at the 8-position is a (D)-p-acyloxy-aamino acid residue, i.e. the residue of an u-amino acid of the (D)-series having an acyloxy group attached atthe P-carbon atom.

Preferred (D)-o-acyloxy-a-amino acid residues are those of formula 11 R2 2 1 R,-CO-0-uii(js) 1 (11) -NH-um-uu- 25 (D) wherein R, is hydrogen, Cl-4alkyl or phenyl and R2 is hydrogen or methyl. 30 Especially preferred cyclosporins in accordance with the present invention are those wherein the amino acid residue at the 8-position is an 0-acyl-(D)-seryl or 0-acyi-(D)-threonyl residue, in particular an 0- acyl-(D)seryl or 0-acyi-(D)-threonyl residue of formula 11 above.

In one group of cyclosporins in accordance with the present invention, the amino acid residue at the 8-position is an 0-acyl-(D)-seryl residue, especially an 0-acyl-(D)-seryl residue wherein the acyl moiety has the formula R, -CO- in which R, has the meaning given above.

In a second group of cyclosporins in accordance with the present invention, the amino acid residue atthe 8-position is a (D)-P-acyloxy-a-amino acid residue, especially an 0-acyi- (D)-seryl residue wherein the acyl moiety has the formula R, -CO- in which R, is hydrogen or Cl-4alkyl, and the residue at the 5-position is an M-norvalyl residue.

Most preferred are cyclosporins of formula 1 X-Y-Sar-MeLeu-Z-MeLeu-Ala-Q-MeLeu-MeLeu-VaI 1 2 3 4 5 6 7 8 9 11 45 -j wherein X is -MeBmt- or -dihydro-MeBmt-, Y is -aAbu-, -Ala-, -Thr-, -Valor -Nva- Z is -Val- or -Nva, and Q is a residue of formula If as defined above.

In formula 1, Q is preferably an 0-acyl-(D)-seryl or 0-acyl-(D)-threonyl residue wherein the acyl moiety has the formula R, -CO- in which R, has the meaning given forformula 11. Y is preferably -aAbu-, -Thr-, -Val- or -Nva-.

A group of cyclosporins in accordance with the present invention are those of formula I as defined above, 55 wherein Y is -uAbu- or -Nva-, Z is -Valand R2 is hydrogen.

A further group of cyclosporins in accordance with the present invention are those of formula I as defined above, wherein Y is aAbu- or -Nva-, Z is -Nva-, R, is hydrogen or C1- 4alkyl and R2 is hydrogen.

The present invention also provides a process for the production of a cyclosporin wherein the amino acid residue at the 8-position is a (D)-acyloxy-u--amino acid residue, for example a (D)-p-acyloxy-a--amino acid residue, e.g. for the production of a cyclosporin of formula I as defined above, which process comprises:

a) Acylating a cyclosporin wherein the amino acid residue at the 8position is a (D)-hydroxy-a-amino acid residue, for example a (D)-phydroxy-a-amino acid residue, e.g. acylating a cyclosporin of formula III 3 -X-Y- Sar- MeLeu -Z- M eLeu -Ala -W- M eLeu - MeLeu - MeVal 1 2 3 4 5 6 7 8 9 10 11 wherein X, Y and Z have the meanings given above for formula land W is a residue of formula IV R2 1 HO-CH 1 -NH-um-uu- (D) GB 2 155 936 A 3 (111) (IV) wherein R2 has the meaning given above for formula 11, to introduce a group R, -CO-, wherein R, has the meaning given above for formula 11, at the p-position of said residue IV; or b) Reducing a cyclosporin wherein the amino acid residue at the 1 -position is -MeBmtand the residue at the 8-position is a (D) -acyloxy-a-amino acid residue, for example a (D)- P-acyloxy-ot-amino acid residue, to produce the corresponding cyclosporin wherein the residue at the 1 - position is -dihydro-MeBmt, e.g. reducing a cyclosporin of formula 1 as hereinbefore defined, wherein X is -MeBmt-, to produce the corresponding cyclosporin wherein X is -dihydro-MeBmt.

Process step a) above may be carried out in accordance with standard procedures for the acylation of hydroxy groups, for example by reaction with (preferably 2 equivalents or, when Y = -Thr-, 1 equivalent) of an appropriate acyi-, e.g. Cl-salkanoyl- or benzoyl-halide, or corresponding -anhydride or, for formylation, by 25 reaction with e.g. acetic-formic anhydride, at a temperature of e.g. from about -10 to WC. The reaction is carried out under anhydrous conditions, suitably in the presence of an inert solvent or diluent such as methylene chloride, and in the presence of a condensation agent such as 4-dimethyi-amino-pyridine. In this connection it is to be noted that the reaction proceeds with acylation occurring at the OH group of the amino acid residue at the 8-position, in preference to the hydroxy group of the amino acid residue at the 1 -position. 30 Process step b) may be carried out analogously to known methods for reducing naturally-occurring cyclosporins to the corresponding dihydrocyclosporins, for example by catalytic hydrogenation, e.g. in accordance with the general methods disclosed in U.K. Patent Specification No. 1,567,201.

Hydrogenation is suitably effected under neutral pH conditions at temperatures of from about 20'to about 300C and at atmospheric or slightly evelated pressure, in the presence of a catalyst such as platinum or, 35 preferably, palladium (e.g. palladium on charcoal) in the presence of an inert solvent or diluent such as ethyl acetate or lower aliphatic alkanols such as methanol or iso-propanol.

Cyclosporins having a p-hydroxy-a-amino acid residue at the 8-position, in particular [(D)Serl'-Cyclosporine and [Dihydro-MeBmtl'-[(D)Serl'-Cyclosporine, suitable for use as starting materials in process step a) above are known and have been described together with processes for their production, e.g. in the aforementioned 40 European patent no. 0 056 782. Other cyclosporins having a hydroxy-ot-amino acid residue at the 8- position and required as starting materials for process step a), may be prepared analogously or in accordance with the general procedures of the cyclosporin total-synthetic method described in European patent no. 0 034 567 to which publication 0 056 782 cross-refers, or in accordance with the procedures hereinunder described in particular in the accompanying examples.

The cyclosporins starting materials for use in process step b) above are obtainable in accordance with the method of process step a).

Although the cyclosporin starting materials of formula Ill above specifically disclosed in the accompanying examples are embraced by the broad disclosure of the aforementioned European patent no. 0 056 782, certain of these cyclosporins are formally novel over the teachings of that publication, i.e. have never previously been described as such. In accordance with the present invention it has also been found that these cyclosporins possess especially interesting or advantageous biological activity or profile, in particular in relation to immunosuppressive activity, and especially in relation to prevention of transplant, e.g. organ transplant, rejection, e.g. as compared with known cyclosporins of formula Ill, i.e. cyclosporins of formula Ill specifically disclosed in European patent no. 0 056 782.

Accordingly in a further aspect the present invention also provides a cyclosporin of formula Ilia.

so 4 GB 2 155 936 A 4 X'-Y'-Sar- MeLeu -Z'- MeLeu -Ala -W'- MeLeu - MeLeu - MeVal 1 2 3 4 5 6 7 8 9 10 11 wherein Y'is -a.Abu-, -Thr-, -Val- or -Nva-, Z'is -Val- or, when Y' is -o.Abu- or -Nva-, -Nva-, Wis -(D)Ser- or, when Y'is -(xAbu- and Z'is -Val-, -(D)Thr-, and S'is -MeBmt- or, when Y'is -Thr-, -Val- or -Nva-, Z'is -Val- and Wis -(D)Ser-, -dihydro-MeBmt-.

Specific cyclosporins of formula [Ha are:

a) [(D)Thrl'-Cyclosporine b) [Thr12- [(D)SerjCyclosporine c) [DihydroMeBmtll-[Thr]2- [(D)SerjCyclosporine d) [Va 112- (D)Serl'-Cyclosporine e) [Dihydro-MeBrnt11-[Va 1]2- [(D)Serl'- Cyclosporine f) [Nval'[(D)Serl'-Cyclosporine 9) [Dihydro-MeBmtll-[NVa 12- [ (D)SerjCyclosporine h) [Nva]5--[(D)Serl'-Cyclosporine, and i) [Nva]2[Nval'-[(D)Serl'-Cyclosporine Ilia) Of the above listed cyclosporins, a), b), e), f) and i), and in particular a), f) and i) are of especial interest, having regard to their activity (e.g. immunosuppressive activity)/activity profile, e.g. in relation to cyclosporins specifically disclosed in European patent no. 0 056 782.

In addition to the foregoing the present invention also provides a process forthe production of a 25 cyclosporin of formula Ilia as defined above, which process comprises:

c) Deprotecting a cyclosporin of formula 111 as defined above which is in 0-protected form; d) Cyclising a straight chain undecapeptide comprising the sequence F -W'-MeLeu-MeLeu-MeVai-X'-Y'-Sar-MeLeu-Z'-MeLeu-Ala -1 9 10 11 1 2 3 4 5 6 7 35 wherein Y', Z', Wand X'have the meanings given above for formula Ilia, said undecapeptide being in unprotected or O-protected form, and when required, carrying out process step c; e) For the production of a cyclosporin of formula Illa wherein Y'is-Thr-, -Valor-Nva-, Z'is -Val- or, when Y'is -Nva-, -Nva-, W' is - (D)Serand X'is -MeBmt-, cultivating a [Thr]2-CyClosporine, [Val]2- CyClosporine [Nva112-CyClosporine or [NVaj2-[Nva]'-Cyclosporine producing fungus strain in contact with a nutrient medium containing (D)-Serine and isolating the cyclosporin of formula [Ila from the obtained culture medium; f) For the production of a Gyclosporin of formula Ilia wherein X' is -dihydro-MeBmt, reducing the corresponding CyClosporin of formula Ilia wherein X' is -MeBmt-. 50 Undecapeptides suitable for use in process step d) above may be obtained analogously to the general methods described in the above mentioned European patent no. 0 056 782, e.g. in relation to the flow chart to Example 1 a thereof, by combination of the pepticle sequence comprising residues 8 through 11 of the Gyclosporin molecule with the sequence comprising residues 1 through 7 but with the required substitution of residues at positions 2 and/or 5 and/or 8. Suitably the -(D)Ser- or - (D)Thr- residue at the 8-position is in O-protected form, e.g. in the form of the O-t-butyl derivative. Cyclisation is carried out using the particular 55 techniques described in the said European Patent, with final removal of O- proteding groups when present [process step c)] in accordance with techniques known in the art of peptide chemistry.

The preferred fungus strain for use in the method of process step e) is the strain NRRL 8044 of the species Tolypocladium inflatum (Gams), a culture of which has been deposited with the United States Department of Agriculture (Northern Research and Development Division), Peoria, Ill., USA and is freely available to the 60 public. Afurther culture of this strain has been deposited with the Fermentation Research Institute, Inage, Chiba City, Japan, underthe code number FRI FERM-p No. 2796. The morphological characteristics of said strain, originally classified as belonging to the species Trichodermapolysporum (Link ex Pers.), as well as methods for the preparation and maintainance of pre- and sub-cultures are fully described e.g. in UK patent specification no. 11491,509.

GB 2 155 936 A 5 In accordance with process step e) the selected strain [e.g. Tolypocladium inflatum (Gams)] is suitably maintained for a period of ca. 2 weeks at a temperature of ca. 27'C in a culture medium such as described in the following examples, in the presence of added (D)- or (D,L)-serine. T" amino acid precursor is suitably added in an amount of from about 1 to about 15 g, more preferably from about 4 to about 10 g/litre culture medium. Suitably the culture medium also contains added amino acid precursor for the residue present in 6 the desired cyclosporin at position 2, e.g. in amounts of from about 6.0 to about 10.0, preferably about 8.0 g/litre culture medium. Following incubation the culture is harvested and the obtained cyclosporin of formula Ilia extracted in accordance with known techniques, e.g. by comminution of conidia and mycelia, followed by extractive and/or absorptive isolation. The initially obtained, raw cyclosporin may thereafter be purified e.g. chromatographically and/or by recrystallisation, in particular to effect separation from other 10 cyclosporin contaminants in particular "natural cyclosporin" contaminants.

Process step f) above may be carried out e.g. using the same methods hereinbefore described in relation to process step b).

The following examples are illustrative of the processes of the present invention.

Example 1 Synthesis of[(O-acetyl)-(D)Ser)9-Cyclosporine[Formula /: X = MeBmt-, Y= -aAbu-, Z = -Val-, 0 -0-acetyl-(D)Ser-1.

20 mg 4-dimethylaminopyridine are added to 47 mg [(D)Ser']-Cyclosporine (prepared in accordance with 20 the method described in Example 1 or 3 of the above mentioned European Patent No. 0 056 782) dissolved in 3 mi methylene chloride. 6.1 mg of freshly distilled acetylchloride in 1 m[ methylene chloride are then added and the obtained reaction mixture is stirred for 1 hour at room temperature. The reaction mixture is diluted with 50 mi methylene chloride and shaken with 30 mi H20. The organic phase is separated, dried over Na2S04, filtered off and evaporated. The residue is filtered on 60 g silica gel (0.062 - 0.20 mm) using 25 methylene chloride/5 % methanol as eluant and collected in 25 mi fractions. The title compound is recovered from fractions 4 to 8 bythin layer chromatography using CHC13/5 % methanol as carrier phase:

[,120= -202' (c = 0.92 in CHC13).

D Example 2

The following compounds may be prepared analogously to example 1 starting from the corresponding non-acylated cyclosporin:

2.1 [(O-benzoyl-(D)Serl'-Cyclosporine [Formula 1; X = -MeBmt-, Y = -(xAbu-, Z = -Val-, Q = -0-benzoyl-(D)Ser-1: [,]20 = D -220' (c = 1.0 in CHC13); O-acetyl-(D)Thr]&CyClosporine [Formula I:X = -MeBmt-, Y = -otAbu-, Z = -Val-, Q = -0-acetyl-(D)-Ser-] [(x]2DO = -219')C = 1.0 IN CHCL3); 2.3 [Nva1'-[O-acetyI-(D)Serj8-CyCI osporine [Formula 1:

X = -MeBmt-, Y = -Nva-, Z = -Val-, Q = -0-acetyl-(D)Ser-1:

[a_12,()= -240'(c = 1.0 in CHC13)/-233'(c = 0.8inCHCI3)/-177'(c= 0.76 in CH30H): m.p. = 143-147'C.

2.4 [Va 112- [0-acetyl-(D)Serl'-Cyclosporine [Formula 1; X = - MeBmt-, Y = -Val-, Z = -Val-, Q = -0-acetyl- (D)Ser-1:

[a]'Dl = -219' (c = 0.9 in CHC13); [Nva]5-[O-acetyl-(D)Ser]8-CyClosporine [Fcrmula 1:

X = -MeBmt-, Y = -uAbu-, Z = -Nva-, Q = -0-acetyl-(D)Ser-1:

[&-12DO = - 215' (c = 1.0 in CHC13); 2.6 [Nva 12 [Nva]5-[O-acetyl-(D)Ser]8-CyClosporine [Formula 1:

X = -MeBmt, Y = -Nva-, Z = -Nva-, Q = -0-acetyl-(D)Ser-1:

[U120 = - 196.9' (c = 1.0 in CHC13); and D 2.7 [I'hrl2-[O-acetyl-(D)Ser]8-CyClosporine [Formula 1: X = -MeBmt-. Y = Thr-, Z = -Val- Q -0-acetyl-(D)Ser-1: (0]20 = -251'(c = 0.86in CHC13)/174'(c= 0.81 in CH30H): m.p. = 143-146'C.

D Example 3 Synthesis of [Dihydro-MeBmt]-[O-acetyl-(D)Ser]'-Cyclosporine [Formula 1: X = -dihydro-MeBmt, Y -(xAbu-, Z = -Val-, Q = -0-acetyl(D)Ser-1:

54 mg of [(O-acetyl)-(D)Ser31-Cyclosporine in 10 ml ethanol are hydrogenated using 10 mg palladium/ charcoal (10 %) at room temperature and under normal pressure. After 20 hours the obtained reaction solution is filtered through a thin layer of talc and the ethanol is evaporated off under vacuum. After further drying under high vacuum, the title compound is obtained:

[0,120 = -205.8'(c = 1.02 in CHC13).

D 6 GB 2 155 936 A 6 Example 4

The following compounds may be prepared either analogously to example 1, starting from the corresponding non-acylated cyclosporin or analogously to example 3, by hydrogenation of the corresponding cyclosporin described in example 2:

4.1 [Dihydro-MeBmt]'-[Nva]2- [0-acetyl-(D)Ser]8-CyClospori ne [Formula 1: X = -dihydro-MeBmt-, Y = -Nva-, Z = -Val-, Q = -0-acetyl- (D)Ser-1:

m.p. = 139 -141'C, [U]20 = 225'(C = 0.88 in CHC13)/-163'(c = 0.76 in CH30H); D [Dihydro-MeBmt]'-[Va 1]2- [0-acetyl-(D)Ser]8-CyClosporine [Formula 1: X = -dihydro-MeBmt-, Y = -Val-, Z = -Val-, Q = -0-acetyl-(D)Ser-1: [0,120 = - 210' (c = 0.85 in CHC13); and D [Dihydro-MeBmt]1JThr]2- [0-acetyl-(D)Ser'-Cyclosporine [Formula 1: X = dihydro-MeBmt-, Y = -Thr-, Z = -Val-, Q = 0-acetyl-(D)Ser-1: [0120 = 241' (c = 1.0 in CHC13)1- 162' (c = 1.0 in CH30H): m.p. = 148 - 150'C. D Preparation of starting materials:

Example 5

The following compounds required as starting materials for the production of the compounds of examples 2.2 through 2.7 may be prepared analogously to the known compound [(D)Serl'-Cyclosporine, the preparation of which is described in Example 1 of European Patent No. 0 056 782, with substitution of the appropriate residues, at positions 2 andlor 5 andlor 8 in the process sequence set forth in the flowchart to 20 Example la of said patent:

5.1 [(D)Thr]B-Cyclosporine [Formula Ilia: X' = -MeBmt-, Y' = -aAbu-, Z' = -Vai-, W' = -(D)Thr-l:

[a]2uOic = -248X (c = 1.0 IN CHCL3); 5.2 [Nval2- (D)Serl'-Cyclosporine [Formula Ilia: X' = -MeBmt-, Y' = -Nva-, Z' = -Vai-, W' = -(D)Ser-l: m.p. = 150 - 153'C; [,120 = D -262'(c = 0.71 in CI-IC13)1-1910 (C = 0.73 in CH301-1); 5.3 [Vai]'-[(D)Serls-Cyclosporine [Formula Ilia: X'= -MeBmt-, Y' = -Vai-, Z' = -Vai-, W' = -(D)Ser-l:

[,]20 = -257' (c = 1.0 in CI-IC13)1- 255' (3 = 0.45 in CI-ICIP- 189' D (c = 0.42 in CH30H): m.p. = 136 - 140'C.

5.4 [Nval'[(D)Ser]8-Cyclosporine [Formula Ill: X'= -MeBmt, Y' = -ctAbu-, Z' = -Nva-, W' = -(D)Ser-l:

[ed2D1 = -212'(c = 1.0 in CHC13); [Nval2- [Nval7- [(D)Serl'-Cyclosporine [Formula Ilia: X'= -MeBmt,Y'= -Nva- ,Z'= -Nva, W' -(D)Ser-Hal2DO = -217' (c = 1.0 in CI-IC13); and 5.6 [Thr]2- [(D)Ser]8-Cyclosporine [Formula Ilia: X'= -MeBmt, Y' = -Thr-, Z' = -Vai-, W' = -(D)Ser-l: [UJ20 = -258' (c = 0.39 in CI- IC13)/-178' (c= 0.40 in CG301-1); D m.p. = 147 - 152'C.

5.5 Example 6

Thecompound of example 5.2 may alternatively be produced microbiological ly as follows:

a) 10 litres of a nutrient medium containing 50 g maltose; 5 g (DL)norvaline; 8 g (D)-serine; 0.75 g KH2P04; 0.5 9 M9S04-71-120; 0.1 CaC12.6H20 and 8 g caseinpeptone per litre are inocuiated with 1 litre of a suspension of conidia and mycelia of the fungus strain NRRL 8044 taken from a 3 day old pre-culture. The incoculated 45 production-medium is filled in 100 mi portions into 100 Erienmeyer flasks which are then incubated for 14 days at27'on an agitator rotating at 180 r.p.m. The mycelium is separated from the culture medium and extracted in a Turrax apparatus by crushing and stirring with 3 x 3 litres of 90 % methanol. The crushed mycefflum is separated from the solvent by suction-filtration and the combined filtrates are concentrated by evaporation under vacuum at a temperature of 4WC until the vapour consists mainly of water alone. The so obtained mixture is extracted 4 x using 0.5 litre 1,2-dichloroethane at each extraction and the combined 1,2-dichloroethane solutions are concentrated by evaporation under vacuum at a temperature of 40'C.

The obtained residue is subjected to gel filtration on Sephadex LH-20 (1. 4 kg; Pharmacia) with methanol, and collected in 280 mi fractions. Fractions 9 - 11, containing a cyclosporin mixture are pooled and then separated by silica gel column chromatography (1 kg of silica gel, granulate size 0.063 - 0.2 mm, "Merck") 55 using water saturated ethyl acetate as eluent (fractions of 500 ml). In accordance with their polarity, (Nva 21-Cyclosporine elutes first (fractions 7 - g), followed by a mixture comprising [Nva 21-[(D)Ser81 Cyclosporine and Cyclosporine. Separation of [Nva 21-[(D)Ser'I- Cyclosporine and Cyclosporine is achieved by silica gel chromatography (280 g, "Merck", 0.63 - 0.2 mm) using chloroform/methanol (98: 2) as eluent (fractions of 100 mi). Fractions 20 - 30, containing crude [Nva 21- [(D)Ser'I-Cyclosporine, are further purified by 60 medium-pressure chromatography on a reversed-phased silica gel column ("Merck" LiChropep RP 18,260 g, granulate size 0.04 - 0.063 mm) with methanollwater (85: 15) as eluent, with collection in 25 m] fractions. The combined fractions 45 - 55 yield pure [Nva 21_[( D)Serll-Cyclosporine as an amorphous white powder.

The pre-culture required for the above process may be obtained as follows:

b) The spore and mycelium suspension used for inoculation is produced from a culture of the originally 65 7 GB 2 155 936 A 7 isolated strain NRRL 8044, cultivated for 21 days at 27'C on an agar medium containing 20 g of malt extract, 20 g of agar, 4 9 of yeast extract per litre of demineralised water. The spores of this culture are taken up in a physiological NaCI solution to give a final concentration of 5 x 106 spores/ml. 10 mi of this suspension are used for inoculation of 1 litre of a nutrient solution having the same combsition as the culture medium of Example 6a, with the exception of the (D)-serine and (DL)-norvaline components, and incubation is effected at 2TC for 3 days on a rotary shaker (200 r.p.m.). This culture is used as inoculum for the producing culture, [Nva 21_ [(D)Sers]- Cyclosporine may be produced on fermenter scale as follows: c) Ca. 109 spores from an agar slant of the strain NRRL 8044 are transferred into a stainless-steel-fermenter containing 20 litres of a pre-culture-medium comprising:

10 Fructose 75 g Amber HC 25 g KH20P4 5 g KCI 2.5 g 15 Dist. Water to 1 litre (p H = 5,5) previously sterilized for 20 minutes at 120T. Favourable incubation conditions area temperature of 27'C, ' airflow of 16 litres per minute at an overpressure of 0.5 bar and stir rotation of 200 r.p.m. The developing 20 pre-culture is incubated for 6 days and 15 litres are then transferred to a stainless steel-fermenter holding 300 litres of production medium comprising:

Maltose 75 9 25 Amber EI-IC 25 g KH2P04 5 I(C1 2,5 g (DL)-norvaline 5 g 0-serine 8 g 30 Dist. Water to 1 litre (p H = 5,5) previously sterilized for 20 minutes at 120'C.

The culture is held at a temperature of 27'C, aerated with 120 litres air per minute at an overpressure of 0.5 35 bar and stirred at 70 r.p.m. Foam control is performed by addition of a silicone emulsion.

After incubation for 14 days the culture, which has a total volume of 275 litres is cooled to 1 O'C and the mycelia removed using a Westfalia separator. The filtrate is extracted by stirring 2 x with ethyl acetate, the extracts are washed with a little water, combined and dried under vacuum. The mycelium is combined with methanol, homogenised and filtered. This extraction is repeated 2 x using 90 % methanol. The methanolic 40 extracts are combined and, with addition of water, concentrated under vacuum. The remaining, aqueous concentrate is extracted 2 x with ethyl acetate, the extracts washed with a little water, combined and concentrated under vacuum. The extracted aqueous phase is re-extracted 2 x with ethyl acetate/isopropanol (8: 2). These extracts are combined and again evaporated under vacuum.

The mycelial and filtrate extracts are filtered using 50 x the amount of Sephadex LH-20 with methanol as 45 eluant, The peak-fractions are then purified chromatographically using 100 x the amount of silica gel 60 (particle size = 0.04 - 0.063 mm) using water-saturated ethyl acetate as eluant. [Nva 21 -Cyclosporine elutes firstfollowed by Cyclosporine and [Nva 2]-[(D)-Ser31-Cyclosporine. These later fractions are subjected to further chromatographic purification using 140 x the amount of silica gel 60 (particle size 0.063 - 0.20 mm) and chloroform/methanol (98: 2) as eluant, to yield pure (Nva 21 - [(D)Ser'Cyclosporine.

so Example 7

The compound of example 5.3 may also be produced microbiologically proceeding analogously to example 6a) but with the following modifications: a) In the nutrient medium - replacement of (DL)-norvaline with 10 9 W-

valine. Following separation of the 55 mycelium from the culture medium extraction as follows:

The crushed mycelium is separated from the solvent by suction-filtration and the combined filtrates are concentrated (under addition of water) by evaporation under vacuum at a temperature of 40'C until the vapour consists mainly of water alone. The obtained mixture is extracted 3 x using 5 litres ethyl acetate at each extraction and the combined ethyl acetate solutions are concentrated by evaporation under vacuum at 60 a temperature of 40'C.

The obtained residue is subjected to gel filtration on Sephadex LH-20 (1. 4 kg; Pharmacia) with methanol.

Those fractions which contain a cyclosporin mixture are pooled and then separated by silica gel column chromatography (3 kg of silicagel, granulate size 0.020 - 0.045 mm, "Grace") using water-saturated ethyl acetate as eluent. In accordance with their polarity, [Va12]_CyClosporine elutes first followed by a mixture 8 GB 2 155 936 A 8 comprising [Va 121-[(D)Ser'I-Cyclosporine asthe majorcomponent. Further purification of [Vaill[(D)Ser'11Cyclosporine is achieved by silica gel chromatography (80 g, "Grace", 0.020 - 0.0045 mm) using acetonelhexane (1: 1) as eluent. Those fractions containing crude [Vai']-[(D)Ser'I- Cyclosporine, are further purified by medium-pressure chromatography on a reversed-phased siiica gel column ("Merck" LiChroprep 5 RP 18,160 9, granulate size 0.4 0.063 mm) with methanollwater (80: 20) as eluent, yielding pure [va 12]-[(D)Ser81-Cyclosporine as a amorphous white powder. b) The pre-culture required is obtained as in example 6 b). [Vaill[(D)Ser'I-Cyclosporine maybe produced on fermenter scale proceeding analogousiyto example 6 c) but with the following modifications:

c) In the production medium -replacement of (DL)-norvaline with 10 g (Qvaline. Following combination of 10 the mycelium with methanol, homogenisation and filtration (repeated 2 x using 90 %methanol) -further processing as follows:

The methanolic extracts are combined and, with addition of water, concentrated under vacuum. The remaining, aqueous concentrate is extracted 3 x with ethyl acetate, the extracts washed with a little water, combined and evaporated under vacuum.

The mycelial and filtrate extracts are filtered using 50 x the amount of Sephadex LH-20 with methanol as eluant. The peak-fractions are then purified chromatographically using 40 x the amount of silica gel 60 (particle size = 0.04 - 0.063 mm) using water- saturated ethyl acetate as eluant. [Va 12]-Cyclosporine elutes firstfollowed by Cyclosporine and [Va 12]-[(D)Ser'I-Cyclosporine. These laterfractions are subjected to further chromatographic purification using 100 x the amount of silica gel 60 and acetonelhexane ( 1: 1) as 20 eluant, and medium-pressure chromatography on reversed-phased silica gel ("Merck" LiChroprep RP 18, granulate size 0.04 - 0.063 mm) with methanollwater (80: 20) as eluant, to yield pure [Va 12]-[(D)Ser'] Cyclosporine.

Example 8

The compound of example 5.6 may also be produced microbiological ly proceeding analogously to example 6 a) but with the following modifications:

a) In the nutrient medium - replacement of (DL-norvaline with 5 g (Qthreonine. Following incubation - extraction as follows:

The mycelium is separated from the culture medium and extracted in a Turrax apparatus by crusing and 30 stirring with 3 x 9 litres of 90 % methanol. The crushed mycelium is separated from the solvent by suction-filtration and the combined filtrates are concentrated (under addition of water) by evaporation under vacuum at a temperature of 40'C until the vapour consists mainly of water alone. The obtained mixture is extracted 3 x using 5 litres ethyl acetate at each extraction and the combined ethyl acetate solutions are concentrated by evaporation under vacuum at a temperature of 4WC.

The obtained residue is subjected to gel filtration on Sephadex LH-20 (2 kg; Pharmacia) with methanol.

Those fractions containing a cyclosporin mixture are pooled and then separated by silica gel column chromatography (2 kg of silica gel, granulate size 0.02 - 0.045 mm, "Grace") using water-saturated ethyl acetate as eluent. In accordance with their polarity, Cyclosporine elutes first, followed by [Q)Ser81 Cyclosporine, followed by [Thr 21_CyCl osporine and finally [Thr2]- [(D)Ser'I-Cyclosporine in crude form.

Further purification of [Thr 21_[(D)Ser'I-Cyclosporine is achieved by silica gel chromatography (50 g, "Grace", 0.02 - 0.45 mm) using acetonelhexane (2: 1) as eluent yielding pure [Thr 2]-[(D)Ser'I-Cyclosporine as an amorphous white powder.

b) The pre-culture required is obtained as in example 6 b).

[Th r 2]-[(D)Ser'J-Cyclosporine may be produced on fermenter scale proceeding analogously to example 6 c) 45 but with the following modifications; c) In the production medium - replacement of (DQ-norvaline with 5 g (Q- threonine. Following incubation and removal of the mycelia using a Westfalia separator - further processing as follows The mycellum is combined with methanol, homogenised and filtered. This extraction is repeated 2 x using 90 % methanol. The methanolic extracts are combined and, with addition of water, concentrated under 50 vacuum. The remaining, aqueous concentrate is extracted 3 x with ethyl acetate, the extracts washed with a little water, combined and concentrated under vacuum.

The mycelial extract is filtered using 50 x the amount of Sephadex LH-20 with methanol as eluant. The peak-fractions are then purified chromatographically using 30 x the amount of silica gel 60 (particle size 0.04 - 0.063 mm) using water-saturated ethyl acetate as eluant. Cyclosporine elutes first followed by [(D)Ser'I-Cyclosporine, followed by [Thr 2]_Cyclosporine and finally [Thr']-[(D)Ser'I-Cyclosporine. These later fractions are subjected to further chromatographic purification using 250 x the amount of silica get 60 (particle size 0.02 - 0.045 mm) and acetone/hexane (2: 1) as eluant, to yield pure [Thr 21[(D)Ser']Cyclosporine.

Example 9

The following compounds, which may be employed as starting materials for production of the compounds of examples 4.1 through 4.3, may be prepared from the indicated cyclosporins of examples 5 through 7, proceeding analogously to example 3.

9 GB 2 155 936 A 9 [Dihydro-MeBrnt11-[Nva]2- f(D)Serl'-Cyc(osporine [Formula Ilia. X' = - dihydro-MaBmt-, Y' = Nva-, Z' = -Vai-, W' = -(D)Ser-1 - prepared from the product of example 5.2 or 6: 20 [a-]D - 251' (c = 1.23 in CI-IC13)/- 179' (c = 1.16 in CH3CH): m.p. = 155 - 157T. [Dihydro-MeBrnt11-[Va 112- [(D)Ser]B-Cyclosporine [Formula Ilia: X' = -dihydro-MeBrnt-, Y' = -Val-, Z' = -Vai-, W' = -(D)Ser-1 - prepared from the product of example 5.3 or 7: 20 1(X1D - 224' (c = 1.0 in CHC13).

9.3 [Dihydro-MeBmtll-[Thrl2-X[(D)Ser]8-Cyclosporine [Formula ffla:

X' = -dihydro-MeBmt-, Y' = -Thr-, Z' = -Vai-, W' = -(D)Ser-1 - prepared from the product of example 5.6 or 8: [UJ20 D - 2621 (c = 0.73 in CHC13)/173' (c = 0.79 in CH30ffi:

m.p. = 156 - 158 'C.

End product cyclosporins, e.g. of formula 1, as hereinbefore defined and described exhibit pharmacological activity as may be shown in the following test methods:

1. Immunosuppressive activity:

1.1 Local haernolysis in vitro in gel [R.I. Mishell and R.W. Dutton, J. Exp. Medicine, 126,423 - 422 (1976)]. 20 Cyclosporins of formula I in accordance with the invention inhibit haernolysis zones compared with untreated controls at concentrations of from 0.01 to 10.0 [tg/ml.

1.2 Lymphocyte stimulation test according to Janossy and Greaves [Clin. Exp. Immunol., 9,483 (1971) and 10, 525 (1972)1:

Cyclosporins of formula I in accordance with the invention inhibit concanavalin A stimulated 25 DNA-synthesis (inhibition of H 3 -thymidine incorporation), cell- proliferation and blastogenesis in mouse-spleen lymphocytes compared with untreated controls at concentrations of from 0.001 to 10.0 I.Lg/ml.

1.3 Mixed lymphocyte reaction [Bach et al., J. Exp. Med. 136,1430 (1972)1:

The reaction (i.e. proliferation and differentiation) of lymphocytes [mouse (Balb/c) spleen cells] on 30 co-incubation for 5 days, with allogenic spleen cells from irradiated mice (CBA) is measured in the presence and absence of test-substance. Reaction in the absence of testsubstance serves as control and is taken as 100%. Reaction in the presence of test-substance is expressed as the % change compared with the 100 % control reaction. Inhibition of reaction is observed using cyclosporins of formula I in accordance with the invention at a concentration of from 0. 001 to 10.0 Lg/ml-'.

1.4 Suppression of organ-rejection:

Kidneys from donor rates (F 344, Y) are transplanted in recipient (WistarFurth, Y) rates.

Test-substance is administered p.o. to recipient rats for 14 days, after which treatment is discontinued.

Test animals are subjected to bilateral nephrectomy seven days after transplant. Since the life of test animals depends on acceptance and functioning of the grafted organ, increase in survival time 40 compared with control animals receiving placebo only serves as a parameter for test-substance efficiency. In the above test method animals receiving cyclosporins of formula I in accordance with the present invention at dosages of from 2.5 to 10 mg/kg p.o. exhibit a survival span from > 60 to: 250 days as compared with untreated controls all of which die as a result of organ rejection within ca. 9 to days.

2. Anti-inflammatory activity Anti-inf lammatory activity may be shown in the adjuvant arthritis test in the rat. For this test adjuvant arthritis is induced according to the method of Pearson and Wood, "Arthr. Rheum" 2,440 (1959).

Cyclosporins of formula I in accordance with the invention are active in this test against developing and 50 established arthritis at doses of from 10to 30 mg/kg/day p.o.

3. Anti-parasitic activity Anti-malaria test according to L. Rane, "Chemotherapy and Drug Resistance in Malaria" ed. W. Peters, Academic Press, New York, 1970. Mice (CF1: male) are infected on day 0 with 0.2 ml of a suspension containing 107 parasitic cells of the species Plasmodium berghei (strain NK 65) administered i.p. Test substance is administered s.c. on day 3, at varying dosages using 5 to 10 mice/dose. The survival time is recorded, and the minimum effective dosage (MED) calculated by comparison of survival time with thatfor untreated controls. For controls, survival time = ca. 7 days. The IVIED is the dosage atwhich survival time is doubled. Cyclosporins of formula I in accordance with the invention are effective in this test at dosages of 60 from 25 to 100 mg/kg/day, s.c.

In view of their immunosuppressive activity, end product cyclosporins e.g. of formula I are indicated for use in the prophylaxis and treatment of diseases and conditions requiring a reduction of the immune response for example to suppress the proliferation of lymphocytes and immunocytes, e.g. in treatment of autoimmune diseases or in preventing the rejection of transplants, e.g. skin, lung, heart, heart-lung, 65 GB 2 155 936 A bone-marrow, kidney, spleen and corneal transplants.

Specific auto-immune diseases for which the cyclosporins of formula I are indicated include all of those for which treatment with Cyclosporine has been proposed or used, for example, aplastic anaernia, pure red cell anaernia, idiopathic thrombocytopaenia, systemic lupus erythematodes7, Oolychondritis, sclerodoma, Wegener granularnatosis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, 5 idiopathic sprue, Crohn's disease, Graves opthalmopathy, sarcoidosis, multiple sclerosis, primary billiary cirrhosis, primaryjuvenile diabetes, uveitis posterior, interstitial lung fibrosis and psoriatic arthritis.

In view of their anti-inflammatory activity, end product cyclosporins, e. g. of formula 1, are also indicated for use in the treatment of inflammatory conditions, in particular inflammatory conditions with an aetiology comprising or including an autoimmue component, e.g. forthe treatment of arthritis and rheumatic diseases 10 such as polyarthritis chronica progrediens.

In view of their anti-parasitic activity, end product cyclosporins, e.g. of formula 1, are also indicated for use as anti-parasitic agents, i.e. for the treatment of parasitic infection of varying type, in particular for the treatment of protozoan as well as trematodal and nematodal parasitic infection. Specific types of parasitic infection include all of those for which treatment with Cyclosporine has been previously proposed in the literature, including schistomosomiasis, filarlasis, feishmania, coccidioidomycosis and in particular malaria.

For the above-mentioned uses as indicated daily dosage is in the range of from about 75 up to about 5,000, preferably up to about 2,000 and most preferably up to about 1,500 mg conveniently administered once or in divided doses 2 to 3x a day. Unit dosage forms, e.g. for oral administration, suitably comprise from about 25 up to about 2,500, preferably up to about 1,000 and most preferably up to about 800 mg cyclosporin of 20 formula I admixed with a pharmaceutically acceptable diluent or carrier therefor.

End product cyclosporins e.g. of formula I may be administered by any conventional route, in particular in accordance with means currently practiced in relation to administration of Cyclosporine, in particular via intravenous infusion e.g. in the case of organ transplant, pre- and immediately post-transplant, as well as during episodes of gastrointestinal disturbance which might otherwise impair absorption, or orally, e.g. in 25 the form of an oral solution.

In accordance with the foregoing the present invention also provides 1. A pharmaceutical composition comprising a cyclosporin of formula I as hereinbefore defined together with a pharmaceutically acceptable diluent or carrier therefor; 2. A Gyclosporin of formula I as hereinbefore defined for use as a pharmaceutical, i.e. for use in treatment 30 by surgery or therapy, in particular for use as an immunosuppressant or anti-inflammatory or anti-parasitic agent; as well as 3. A method of inducing immunosuppression, of treating inflammation or of treating parasitic infection, in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a cyclosporin of formula I as hereinbefore defined.

As hereinbefore described the cyclosporins of formula Ilia are also novel and, in addition to their utility as intermediates, exhibit a pharmacological activity and/or profile, in particular in relation to immunosuppres sive activity and especially in relation to utility in the prevention of transplant rejection, which renders them of especial interest, e.g. in relation to other cyclosporins specifically disclosed in European patent no. 0 056 782. Pharmacological activity of cyclosporins of formula - Ilia may be shown e.g. in the above described test 40 methods 1.1, 1.2,1.3,2 or 3. Thus cyclosporins of formula Ilia in accordance with the present invention are active.

In test 1.1 above at concentrations of from 0.01 to 10 gglmi; in test 1.2 above at concentrations of from 0.001 to 10 tig/mi; in test 1. 3 above at concentrations of from 0.001 to 10 [Lg/M1; in test 2 above at doses from 10to 30 mglkg/day p.o.; and in test 3 above at dosages of from 50 to 100 mglkg/day s.c.

In view of their immunosuppressive activity cyclosporins of formula Illa are indicated for use in the 50 prophylaxis and treatment of diseases and conditions requiring a reduction of the immune response, e.g. for the suppression of proliferation of lymphocytes and immunocytes, e.g. in the treatment of auto-immune diseases, for example in the treatment of specific auto-immune diseases hereinbefore recited in relation to the utility of cyclosporins of formula I or in preventing the rejection of transplants, for example of the various specific types hereinbefore recited in relation to the utility of cyclosporins of formula 1. In view of their anti-inflammatory activity, cyclosporins of formula Ilia are also indicated for use in the treatment of inflammatory conditions, in particular inflammatory conditions with an aetiology, comprising or including an auto-immune component, e.g. forthe treatment of arthritis and rheumatic diseases such as polyarthritis chronica progrediens.

In view of their anti-parasitic activity, cyclosporins of formula Ilia are also indicated for use as anti-parasitic 60 agents, i.e. forthe treatment of parasitic infection of varying type, in particular as hereinbefore described in relation to the utility of cyclosporins of formula 1.

For the above mentioned uses an indicated daily dosage is in the range of from about 75 to about 5,000 mg conveniently administered once or in divided doses 2 to 3x a day. Unit dosage forms, e.g. for oral administration, thus suitably comprise e.g. from about 25to about 2,500 mg cyclosporin of formula Ilia 65 11 GB 2 155 936 A 11 admixed with a pharmaceutically acceptable diluent or carrier therefor.

A suitable daily dosage for any specific cyclosporin of formula Ilia in any particular indication will of course depend in particular on its relative potency of activity in relation to the indication, e.g. condition to be treated. The preferred cyclosporins of formula [Ila are The cyclosporins of formula Illa may be administered by any conventional route, in particular in accordance with means currently practiced in relation to administration of Cyclosporine, in particular via intravenous infusion, e.g. in the case of organ transplant, pre- and immediately posttransplant, as well as during episodes of gastrointestinal disturbance which might otherwise impair absorption, or orally, e.g. in the form of an oral solution.

In accordance with the foregoing the present invention also provides:

1. A pharmaceutical composition comprising a cyclosporin of formula Ilia as hereinbefore defined together with a pharmaceutically acceptable diluent or carrier therefor.

2. A cyclosporin of formula Illa as hereinbefore defined for use as a pharmaceutical, i.e. for use in treatment by surgery or therapy, in particular for use as an immunosuppressant or anti-inflammatory or anti-parasitic agent, as well as 3. A method of inducing immunosuppression, of treating inflammation or of treating parasitic infection, in a subject in need of such treatment, which method compounds administering to said subject an effective amount of a cyclosporin of formula Illa as hereinbefore defined.

Claims (27)

1. A cyclosporin wherein the amino acid residue at the 8-position is a (D)-acyloxy-ot-amino acid residue.

2. A cyclosporin according to claim 1 wherein the amino acid residue at the 8-position is a (D)-p-acyloxy-a-amino acid residue.

3. A cyclosporin according to claim 2 wherein the amino acid residue at the 8position is an 0-acyl-(D)-seryl residue.

4. A cyclosporin according to claim 2 or 3 wherein the amino acid residue at the 5-position is an (L)-norvalyl residue.

5. A cyclosporin according to claim 2 wherein the amino acid residue at the 8-position is an 0-acyl-(D)-threonyl residue.

6. Acyclosporin according to claim 2of formula 1 X-Y-Sar-MeLeu-Z-MeLeu-Ala-G-MeLeu-MeVaI 1 2 3 wherein so (1) 4 5 6 X is -MeBmt- or -dihydro-MeBmt-, Y is -(xAbu-, -Ala-, -Thr-, -Val- or - Nva-, Z is -Val- or -Nva-, and Q is a residue of formula 11 R2 1 IR,-CO-O-CH 1 -INH-Uh-UU(D) wherein R, is hydrogen, Cl-4alkyl or phenyl and R2 is hydrogen or methyl.

7. A cyclosporin according to claim 6 wherein Y is -ocAbu- or -Nva-, Z is -Val- and R2 is hydrogen.

8. a cyclosporin according to claim 6 wherein Y is -aAbu-, Z is -Nva-, R, is hydrogen or Cl-4alkyl and R2ishydrogen 7 8

9 11 12 GB 2 155 936 A 9. Acyclosporin according to claim 1 which is [0-acetyi-(D)-Serl'Cyclosporine.

10. Acyclosporine according to claim 1 which is [Nval2- [0-acetyi(D)Serl'3-Cyclosporine.

11. Acyclosporin according to claim 1 selected from the group consisting of: [Dihydro-MeBmtll-[0-acetyl-(D)Ser]8-Cyclosporine; [0-benzoyi-(D)Ser]8Cyclosporine; and [Dihydro-MeBmtjl-[Nval2-[0-acetyi-(D)Ser]8-Cyclosporine.

12. Acyclosporin according to claim 1 which is [Nva]5- [0-acetyi-(D)SerllCyclosporine.

13. Acyclosporine according to claim 1 selected from the group consisting of:

[0-acetyl-(D)Thrl'-Cyclosporine; [Va 112- [0-acetyi-(D)Serl'-Cyclosporine; [Dihydro-MeBmtj'-[Vail'[0-acetyl-(D)Serl'-Cyclosporine; [Nval2- [Nva]5[0-acetyi-(D)Ser]8-Cyclosporine; [Thrl'[0-acetyi-(D)Ser]8-Cyclosporine; and [Dihydro-MeBmtll-[Thrl2- [0-acetyl-(D)Serl'-Cyclosporine.

14. Process for the production of a cyclosporin wherein the amino acid residue at the 8-position is a (D)-acyloxy-a--amino acid residue, for example a (D)-P-acyloxy-a-amino acid residue, e.g. for the production of a cyclosporin of formula 1 X-Y-Sar-MeLeu -Z- MeLeu -Ala -Q- MeLeu - MeLeu MeVal 1 2 3 4 5 6 7 8 9 10 11 wherein X is -MeBmt- or -dihydro-MeBmt-, Y is -aAbu-, -Ala-, -Thr-, -Val- or -Nva-, Z is -Val- or -Nva- and 30 Qis a residue offormula 11 R2 1 Fil-CO-O-CH 1 -NH-CH-CO- (D) wherein R, is hydrogen, Cl-4alkyl or phenyl and R2 is hydrogen or methyl, which process comprises:

(1) (11) a) Acylating a cyclosporin wherein the amino acid residue at the 8- position is a (D)-hydroxy-(x-amino acid residue, for example a (D)-Phydroxy-ot-amino acid residue, e.g. acylating a cyclosporin of formula Ill so 12 so X-Y-Sar-MeLeu-Z-MeLeu-Ala-W-MeLeu-MeLeu-MeVal- 1 2 3 4 5 6 7 8 9 10 11 wherein X, Y and Z have the meanings given above and W is a residue of formula IV R2 1 Mu- LM 1 -Nti-CM-CO- (D) (IV) 13 GB 2 155 936 A 13 wherein R2 has the meaning given above, to introduce a group R1-CO-, wherein R, hasthe meaning given above, at the (3-position of said residue IV; or b) Reducing a cyclosporin wherein the amino acid residue atthe 1-position is -MeBmt- and the residue at the 8-position is a (D)-acyloxy-a-amino acid residue, for example a (D)-p:acyloxy-a-amino acid residue, to produce the corresponding cyclosporin wherein the residue at the 1- position is -dihydro-MeBmt-, e.g. reducing a cyclosporin of formula I as defined above wherein X is -MeBmt-, to produce the corresponding cyclosporin wherein S is -dihydro-MeBmt-.

15. A pharmaceutical composition comprising a cyclosporin as defined in any one of claims 1 to 13 together with a pharmaceutically acceptable diluent or carrier therefor.

16. A cyclosporin as defined in anyone of claims 1 to 13 for use as a pharmaceutical.

17. A cyclosporin as defined in claim 16 for use as an immunosuppressant, or anti-inflammatory or anti-parasitic agent.

18. A method of inducing immunosuppression, of treating inflammation orof treating parasitic infection in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a cyclosporin as defined in any one of claims 1 to 13.

19. Acyclosporin of formula Ilia, X'-Y-Sar-MeLeu-Z'-MeLeu-Ala-W'-MeLeu-MeLeu-MeVaI 1 (ilia) 1 2 3 4 5 6 7 8 9 10 11 wherein Y' is -a.Abu-, -Th r-, -Val- or -Nva-, Z' is -Val- or, when Y' is -a.Abu- or -Nva-, -Nva-, W' is -(D)Ser- or, when Y' is -(xAbuand Z' is -Val-, -(D)Thr-, and X'is -MeBmt- or, when Y'is -Thr-, -Val- or -Nva-, Tis -Val- and Wis - (D)Ser-, -dihydro-MeBmt.

20. A cyclosporin according to claim 19 which is [(D)Thrl'-Cyclosporine.

21. Acyclosporin according to claim 19 which is [Nva 12- [(D)-Serl'Cyclosporine.

22. Acyclosporin according to claim 19 which is [Nva 12- [Nvail'[(D)Serl'-Cyclosporine.

23. Process for the production of a cyclosporin of formula Illa X'-Y'-Sar-MeLeu-Z'-MeLeu-Ala-W'-MeLeu-MeLeu-MeVal- (ilia) 1 1 2 3 4 5 6 7 8 9 10 111 wherein Y' is -aAbu-, -Thr-, -Val- or -Nva-, Z' is -Val- or, when Y' is - (xAbu- or -Nva-, -Nva-, W' is -(D)Ser- or, when Y' is -uAbuand Z' is Nak, -(D)Thr-, and X'is -MeBmt- or, when Tis -Thr-, -Val- or -Nva-, Tis -Val- and Q is - (D(Ser-, -dihydro-MeBmt-, which process comprises: c) Deprotecting a cyclosporin of formula Ila as defined above which is in O-protected form; d) Cyclising a straight chain undecapeptide comprising the sequence -W'-MeLeu-MeLeu-MeVai-X'-Y'-Sar-MeLeu-Z'-MeLeu-Ala- 8 9 10 11 1 2 3 4 5 6 7 wherein Y', Z', W' and X' have the meanings given for formula Ilia, said undecapeptide being in unprotected or O-protected form and, when required, carrying out process step a; e) For the production of a cyclosporin of formula Illa wherein Y' is-Thr-, - Val- or -Nva-, Z'is -Val- or, when Y' is -Nva-, -Nva-, Wis -(D)Ser- and X'is-MeBmt-, cultivating a [Thr]2-Cyclosporine, [Va 1]2-C yclosporine, [Nva]2-CyClosporine or [Nva]2[Nva]5-CyClosporine producing fungus strain in contact with a nutrient medium containing (D)-Serine and isolating the cyclosporin of formula Illa from the obtained culture medium; f) For the production of a cyclosporin of formula Illa wherein X'is -dihydro-MeBmt, reducing the corresponding cyclosporin of formula Illa wherein X' is -MeBmt-.

14 GB 2 155 936 A 14

24. A pharmaceutical composition comprising a cyclosporin as defined in anyone of claims 19 to 22 together with a pharmaceutically acceptable diluent or carrier therefor.

25. A cyclosporin as defined in anyone of claims 19 to 22 for use as a pharmaceutical.

26. A cyclosporin as defined in claim 26 for use as an immunosuppr6s'sant, or anti-inflammatory or 5 anti-parasitic agent.

27. A method of inducing immunosuppression, of treating inflammation or of treating parasitic infection in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a cyclosporin as defined in any one of claims 19 to 22.

Printed in the UK for HMSO, D8818935, 8185, 7102. Published by The Patent Office, 25 Southampton Buildings, London, WC2A 1AV, from which copies may be obtained.

1